β-Glucuronidase

Our β-Glucuronidases are routinely used for the enzymatic hydrolysis of glucuronides from urine^1,2, plasma^3,4, and other fluids⁵ prior to analysis by enzyme immunoassay, mass spectrometry, gas chromatography, high performance liquid chromatography, or other means. Typically, between 1 and 20 units of glucuronidase is used per µl of plasma, urine, or bile for the enzymatic hydrolysis of glucuronides present in these samples.^1–5 The exact amount needed will depend on the specific conditions used and must be determined empirically.

The enzyme from patella vulgata is reported to be much more effective in hydrolyzing opiod-glucuronides.^6,7 Whereas the Helix pomatia and E. coli enzymes are reported to be slightly more effective in hydrolyzing steroid-glucuronides.⁸

New Technical Article Comparing Performance of Different Enzymes

Learn More about recent application data generated by R&D to optimize hydrolysis for different drug classes using enzymes from different sources and the use of a chromatographicaly purified enzyme to reduce the effect of esterase activity resulting in conversion of 6-MAM to Morphine.

On this page:

• β-Glucuronidase
• Molluskan Source β-Glucuronidases
• Bovine Liver Source β-Glucuronidases
• E. coli Source β-Glucuronidases
• Inhibitors
• Substrates
• References

Glucuronidation, conjugation with glucuronic acid, by the human UDP-glucuronosyltransferase (UGT) family of enzymes plays an important role in the metabolic fate of many drugs and other xenobiotics. This biosynthetic reaction also has a role in the conjugation and excretion of endogenous substrates, such as steroids, bilirubin, and bile acids.⁹ UGT activity results in the conjugation of glucuronic acid to substrates containing sulfhydryl, hydroxyl, aromatic amino, or carboxylic acid moieties. The glucuronides formed are more polar (water soluble) than the parent organic substrate and are generally excreted through the kidney.

β-Glucuronidase (EC 3.2.1.31) catalyzes the reaction:

β-D-glucuronoside + H2O <--> D-glucuronate + an alcohol

One Sigma or modified "Fishman" unit will liberate 1.0 µg of phenolphthalein from phenolphthalein glucuronide per hr at 37°C at pH 5.0 (pH 6.8 for the E. coli source) (30 min assay).

Molluskan Source β-Glucuronidases

β-Glucuronidase preparations isolated from mollusks also contain sulfatase activity. For this reason, the sulfatase activity of these preparations is also reported.

One unit of sulfatase will hydrolyze 1.0 mmole p-nitrocatechol sulfate per hr at pH 5.0 at 37 °C.

Bovine Liver Source β-Glucuronidases

Bovine β-Glucuronidase is a 290 kD protein with an isoelectric point of 5.1.¹⁰

Bovine preparations typically contain small amounts of sulfatase activity, usually less than 0.5%.

E. coli Source β-Glucuronidases

β-Glucuronidase is a ~290 kDa tetrameric protein with an isoelectric point of 4.8.¹¹ Unlike the enzyme preparations from mollusks that naturally contain β-glucuronidase and sulfatase activities in almost equal amounts, the preparation of β-glucuronidase from E. coli is essentially free of sulfatase activity. The enzyme from E. coli has a high rate of hydrolytic activity and it retains this activity during hydrolysis better than similar enzymes that are more sensitive to changes in the concentration of β-glucuronide conjugates. The enzyme preparation from E. coli has been shown to be useful for determining the presence of androsterone, 17-hydroxycorticosteroids, and estriol in urine.¹² The E. coli enzyme has also been shown to be more active against estrogen conjugates than other sources of the enzyme.¹³

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References

1. Xu, X., et al., J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 780, 315-330 (2002).
2. Staimer, N., et al., Anal. Chim. Acta, 444, 27-36 (2001).
3. Zhai, P., et al., Am. J. Physiol. Heart Circ. Physiol., 281, H1223-H1232 (2001).
4. Anderson, A. et al., Cancer Chemother. Pharmacol., 44, 422-426 (1999).
5. Nobilis, M., J. Chromatogr. A., 1031, 229-236 (2004).
6. Combie, J. et al., Clin. Chem., 28, 83-88 (1982)
7. Combie, J. et al., Res. Commun. Chem. Pathol. Pharmacol., 35, 27-41 (1982)
8. Wakabayashi, M., et al., J. Biol. Chem., 236, 996-1001 (1960)
9. Tephly, T.R., et al., Adv. Pharmacol., 42, 343-346 (1998).
10. Himeno, et al., J. Biochem.(Tokyo), 76, 1243 (1974)
11. Kim, D-H, et al., Biol. Pharm. Bull., 18, 1184-1188 (1995).
12. Graef, V., et al., Clin. Chem., 23, 532-535 (1977).
13. Jayle, M.F., Scholler, R., Jarrige, P. & Mtay, S. Bull. Soc. Chim. Biol. 41, 1593. (1959)